Tissue cell culture is therefore carried out on solid continuous surfaces. On laboratory scale these are in the form of petri-dishes, flat sided bottle or roller bottles. Recent improvements, applicable also to large scale culture have been the development of spheres of various sizes such as microbeads, as well as of ceramic blocks traversed by small rectangular channels. In all these devices the cells adhere and propagate on the available surface of a solid. Cells will attach only to surfaces that have suitable surface compositions and an electrical surface charge. Materials that have proved successful for this purpose are glass, ceramics, polyvinyl chloride, polystyrene, dextran, collagen, and certain other materials. Frequently the cell attachment surfaces of such materials are treated with 95% sulfuric acid, or preferably with a corona or oxygen plasma discharge to improve cell adherence. Under culturing conditions such as those described in (Kruse & Patterson Tissue Culture--Methods and Applications, Academic Press 1983), cells coming in contact with these surfaces will attach themselves to the surface, and reproduce until they cover the surfaces in a contiguous, confluent monolayer. On reaching confluence cell growth ceases. The phenomenon of cell growth cessation when a contiguous layer of cells is achieved is called "contact inhibition" and is a characteristic of non-cancerous cell growth on two dimensional surfaces.
The growth surfaces used at present for in vitro tissue culture suffer from certain serious shortcomings:
a) They provide limited area per unit volume of nutrient medium supplied, so that the number of cells that can be grown per unit volume of nutrient medium is as much as three orders of magnitude smaller than the volumetric densities of cells in in vitro tissue.
b) They require high concentrations of inoculated cells in order to initiate cell growth. Typically cells are introduced to a fresh culture surface at a concentration and number which is 20-30% of the final number reached at confluence and it is usually impossible to inoculate with less then 10% of the number reached at confluence.
c) Cell growth of non-cancerous cells limited to monolayers;
d) Cells tend to detach from their anchorage surface under conditions that include: monolayer saturation; high or low serum concentration; medium depletion, and particularly, viscous shear caused by stirring or perfusion of the culture medium. Cell growth and biosynthesis stop on cell detachment.
Though non cancerous cells are grown on two dimenisonal surfaces, it has long been known that normal cells will also grow in gels, and specifically those of collagen. One such gel is available commerically (trade name "Gelfoam", Upjohn, Kalmazoo). Gels have however a poor dimensional stablility and, due to their internal structure are unsuitable for growing cells to a reasonable density and especially not for systems with medium perfusion. They have therefore not found use for the large scale culture of cells. Their use is limited to laboratory studies of cell morphology, or studies of the "hystotypic" growth of primary cultures (e.g. Dougles et al., In Vitro, 16, 306-312 (1980)).
Also relevant to this invention is the fact that certain cells and particularly cancerous mammalian cells and altered forms of cells such as hybridomas can be grown freely suspended in culture medium or immobilized in small beads of porous gelled material such as carrageenan, agarose, collagen, gelatin and similar starch or protein or polymer materials. Cells may be mixed into such gels prior to casting the gels into the form of small beads or particles that will be suspended in nutrient medium. (Karkare, S. B., Phillips, P. G., Burke, D. H., and Dean Jr., R. C. "Continuous Production of Monoclonal Antibodies by Immobilized Hybridoma Culture" A.C.S. Annual Meeting Phila. Pa., Aug. 27, 1984, available from Verax Corp. Hanover, N.H.).
Alternatively, cells may be immobilized in microcapsules as described by Lim et al., Microencapsulation of Living Cells and Tissues; J. Pharm. Sci., 70, 4: 351-354, April 1981; Nilsson., Entrapment of Animal Cells for Production of Monoclonal Antibodies and other Biochemicals, Nature 302, 629-630, 1973.
The invention herein described also relates to the cultivation of such non-anchorage dependent cells and other cells whose growth and harvesting may be facilitated by some form of immobilization as described above.